There has been considerable progress in the past few years on understanding the structure and function of T cell receptors (TCR). The structures of several alpha, beta TCR: peptide/MBC complexes have recently been solved and binding studies have shown that these interactions, and those of TCR Vf3 regions with enterotoxins, exhibit low affinities. Various models have predicted that lower affinities and/or faster off-rates might be necessary for optimal T cell signaling. However, because TCRS with higher affinities and long off-rates have not been available, it has not been possible to test such models. We have developed a yeast display technology to produce stabilized, higher affinity TCRS that will allow us to test such predictions. Our work has also used alanine scanning mutagenesis of the TCR to produce energy maps of the TCR:pMHC interface. This information, together with the crystal structure of a TCR:pMHC complex (2C TCR:SIYRJKb and a model of the 2C TCR:QL9/Ld complex) showed that the majority of binding energy is directed at the MHC helices, not at the peptide, in both allo- and self-MHC interactions. However, it is still not clear to what extent different regions of the TCR could contribute to peptide specificity and to what extent the TCR might detect peptide-induced changes in the MHC. Finally, we have recently observed that the contribution of CD8 to pMHC binding by the 2C TCR can vary quite significantly, depending on the pMHC ligand. The explanation for this remains unknown. The yeast display system and the higher affinity TCRs provide strategies for examining many of these questions that relate to the structure and function of the TCR complex in T cell activation. The project will use the 2C T cell system to: 1) isolate higher affinity TCR variants in vitroby yeast display in order to provide insights into which TCR regions confer peptide versus MHC specificity and the extent of cross-reactivities that might be expected among TCR, 2) determine the effects of TCR affinity and kinetics on T cell function by transfecting selected TCR variants into T cells, followed by T cell activity assays, 3) examine the quantitative roles and mechanism of action of CD8a and CD8B in pMHC recognition and T cell function, 4) explore the molecular basis of affinity by solving the structures of TCRs that have high affinity for pMHC and enterotoxin SEC3, and 5)continue our efforts in the development of soluble, high affinity TCR as potential diagnostic and therapeutic agents. The latter studies will include further use of these TCR to improve the sensitivity in detection of pMHC on antigen-presenting cells and as immunosuppressants of T cell activation.